Characterization of the tertiary structure of the peptidoglycan of Enterococcus faecalis

Solid-state NMR spectra of whole cells and isolated cell walls of Enterococcus faecalis grown in media containing combinations of ¹³C and ¹⁵N specific labels in d- and l-alanine and l-lysine (in the presence of an alanine racemase inhibitor alaphosphin) have been used to determine the composition and architecture of the cell-wall peptidoglycan. The compositional variables include the concentrations of (i) peptidoglycan stems without bridges, (ii) d-alanylated wall teichoic acid, (iii) cross-links, and (iv) uncross-linked tripeptide and tetra/pentapeptide stems. Connectivities of l-alanyl carbonyl‑carbon bridge labels to d-[3-¹³C]alanyl and l-[ε-¹⁵N]lysyl stem labels prove that the peptidoglycan of E. faecalis has the same hybrid short-bridge architecture (with a mix of parallel and perpendicular stems) as the FemA mutant of Staphylococcus aureus, in which the cross-linked stems are perpendicular to one another and the cross-linking is close to the ideal 50% value. This is the first determination of the cell-wall chemical and geometrical architecture of whole cells of E. faecalis, a major source of nosocomial infections worldwide.